L-threonine, a kind of essential amino acid, is widely used as an additive to animal feed and food, and as fluids and synthetic materials for medical and pharmaceutical use. L-threonine is produced by fermentation using synthetic mutants derived from wild types of Escherichia Coli, Corynebacterium, Serratia, and Providencia. These mutants are known to include amino acid analogs- and pharmaceutical-resistant mutants and synthetic mutants thereof rendered auxotrophic for diaminopimelic acid, methionine, lysine, or isoleucine (Japanese Laid-open Patent Application No. hei 2-219582, Appl., Microbiolo. Biotechnol., 29, 550-553 (1988), and Korean Patent Publication No. 92-8365). Korean Patent Application No. 90-22965 discloses the L-threonine-producing strain TF4076 (KFCC10718) that is auxotrophic for methionine and resistant to threonine analogs (AHV: α-amino-β-hydroxyvaleric acid), lysine analogs (AEC: S-(2-aminoethyl)-L-cysteine), isoleucine analogs (α-aminobutyric acid), and methionine analogs (ethionine).
A common approach to increase the level of expression of a particular gene uses a plasmid that gives a greater copy number to a microorganism in order to increase the number of genes in the microorganism (Sambrook et al., Molecular cloning, Second Edition, 1989, 1.3-1.5). A target gene is integrated into a plasmid, and the host microorganism is transformed with the recombinant plasmid to cause an increase in the number of genes in the host microorganism according to the copy number of the plasmid. A partial success in this type of approach to improve threonine productivity is reported in U.S. Pat. No. 5,538,873. However, most technologies using such recombinant plasmids overexpress a particular gene, which is undesirable for the host microorganism, and causes a problem of plasmid instability so that the plasmid may be lost during cultivation of the recombinant strain.
To address this problem, approaches to add antibiotics to culture media or to use an expression regulatory plasmid were suggested (Sambrook et al. Molecular cloning, Second Edition, 1989, 1.5-1.6 & 1.9-1.11). In the approach of using the expression regulatory plasmid to yield a particular product, cell cultivation is performed under non-expression conditions in the growth stage to reduce disadvantages to the host microorganism and transient expression is induced after full growth of the microorganism. However, most of these expression regulatory plasmids may be used only in the case that the final product is protein.
Producing primary metabolites is closely associated with the growth of microorganisms, so it is difficult to increase the yield of the primary metabolites unless target genes are expressed in the growth stage. The production of threonine, a primary metabolite, is such a case.
As an effort to compensate for this drawback, a particular threonine biosynthetic gene was incorporated into a chromosomal DNA to produce threonine (U.S. Pat. No. 5,939,307). However, this approach replaces a chromosomal gene by an inducible promoter-substituted gene, which is hardly expected to markedly increase the expression of the threonine operon gene.
Therefore, the present inventors completed the present invention by inactivating threonine dehydratase (tdc) gene in the chromosome, which involves in one of the threonine degradation pathways, to inhibit the degradation of threonine while the other inherent generic function of the host microorganism remains and thus increase the yield of threonine.
Most current genetic engineering techniques applied to increase the yield of threonine are focused on the biosynthetic pathway starting with oxaloacetate. However, the present invention involves also the activity of threonine dehydratase (tdc) gene in a threonine degradation pathway to efficiently and markedly increase L-threonine yield.